D-tagatose is a monosaccharide that has sweetness equal to about 90% of that of sugar while having properties, including low calorie and non-carious properties. It can be used as a healthy sweetener without causing various adult diseases, unlike conventional sweeteners.
Due to such properties, D-tagatose is receiving as a substitute for sugar and is known to have high market potential in the food market. However, tagatose is a rare sugar that is not abundantly present in nature, but is contained in milk products or some plants in very small amounts. For this reason, in order for tagatose to be used as a low-calorie functional sweetener, technology capable of producing tagatose should be developed.
D-tagatose was produced by a chemical isomerization process from D-galactose using Ca(OH)2 as a catalyst by Arla Foods Ingredients Inc. in 2003, and has been marketed under the brand name “Gaio-tagatose”. However, it is known that the chemical isomerization process is excellent in terms of isomerization conversion yield, but has shortcomings in that recovery and purification are difficult and the process is complex, and thus the total yield of the process is lower than that of an enzymatic isomerization process.
L-arabinose isomerase (EC 5.3.1.5) is an enzyme that catalyzes an isomerization reaction of converting L-arabinose into L-ribulose. In addition, it is known that L-arabinose isomerase converts not only L-arabinose (that is the natural substrate thereof) into L-ribulose, but also D-galactose (that is a substrate structurally similar to L-arabinose) into D-tagatose.
The most important factor capable of contributing to an increase in the productivity of a process of producing D-tagatose from D-galactose using L-arabinose isomerase is to develop an enzyme, which has good reactivity and can be successfully applied to the production process, through the modification of isomerase. Because an increase in productivity plays a crucial role in the maximization of profit by a decrease in production cost and the success of business, there has been a continued need to modify arabinose isomerase.
Arabinose isomerase from Thermotoga neapolitana DSM 5068 that is a thermophilic microorganism has a very high thermal stability, but needs to be further improved in order to ensure the economic productivity of arabinose isomerase, which is comparable to that of glucose isomerase.
Generally, methods of producing variant enzymes to increase the activities of enzymes or make enzymes active for new substrates are largely divided into a random mutagenesis method and a rational design method. The random mutagenesis method is widely used, because it can be used without requiring special information about a target enzyme. However, it requires a screening system capable of processing a very large number of variant enzymes. On the other hand, the modification of enzymes by rational design requires no special screening system, because it produces only a limited number of variant enzymes. However, in the case of rational design, factors that determine the catalytic mechanism, substrate binding property or substrate specificity of a target enzyme should be investigated in detail.